The present invention relates to adjustable height and width apertures for on-column capillary detector cells used for capillary methods of separation, and in particular to adjustable height and width apertures for on-column photo-detector cells for capillary zone electrophoresis (CZE), also known as high-performance capillary electrophoresis (HPCE).
Electrophoresis is a separation technique in which the fractionation of the components of a mixture is achieved by the migration of the components through a solution under the influence of an electric field. The individual components move through the solution at varying rates in response to the electric field's influence. The differences in migration rates are generally a function of the charge and volume of a component. Electrophoresis is the dominant separation method used in the study of DNA, proteins and other biological substances.
Similar to chromatography, the term "theoretical plate number" is used to describe the separation efficiency of electrophoresis. As the number of theoretical plates a mixture passes through increases, the degree of separation of the mixture's components consequently increases. As the number of theoretical plates a mixture passes through over a unit period of time increases, the rate of separation of the mixture's components consequently increases.
CZE is a recently developed form of electrophoresis in which a sample solution is introduced into a fine tube (50-75 micron inside diameter) filled with a buffered liquid with separation occurring as a result of the differential movement of sample components toward one of two electrodes by which an electric field is applied to the contents of the tube. Some major problems relative to electrophoresis methods, such as dissipation of heat and suppression of convection, have been greatly improved by using fine capillary tubes. CZE can generate 10.sup.5 to 10.sup.6 theoretical plates within 30 minutes. This separation technique has been successfully applied to analyze a variety of samples including proteins, amino acids, nucleosides, inorganic ions and neutral molecules.
Until the present invention, suitable means for detecting sample components resolved by CZE have not been well developed. The problem is that for a typical CZE peak having a retention time of 500 seconds and 250,000 theoretical plates, the peak width is four seconds. In a capillary with a 50 micron inside diameter and a linear velocity of one mm per second, four seconds corresponds to only eight nanoliters in volume. Stated another way, CZE requires the detection of a series of segments of separated sample components occupying at best 4 mm long segments of the capillary so that the sample volumes to be tested for the presence of the component is at best several nanoliters. Making matters more difficult, the segments will only be separated by one mm or even less and the series of separated sample component segments will be passing through the capillary at a rate of one mm a second. To prevent overlap in the detection of the segments, a detector cell is required having small volume and high sensitivity.
On-column detectors have been used to meet these requirements, employing fluorescent, electrochemical, ultraviolet (UV) and visible (VIS) absorption spectrophotometric detection methods, such as those methods disclosed in Walbroehl et al., J. Chromatogr., 315, 135 (1984) and Terabe et al., Anal. Chem., 56, 111 (1984). UV detectors, though less sensitive than fluorescence detectors, are still the most widely used because of their relative versatility. A section of the capillary downstream of the region where separation occurs is passed through a spectrophotometer detector cell. Light is transmitted through the capillary, and the sample component is identified by its characteristic absorption pattern.
While it has been possible in the past to construct on-column UV detector cells, the prior art was not successful when reducing cell volume to maintain detector sensitivity. This occurs when a light beam having a thickness dimension greater than the inside diameter of the capillary is transmitted through the capillary. The light passing outside of the inner diameter of a capillary creates a high background and drowns out the absorption signal.
Moreover, the prior art did not set a reasonable width of the light beam to avoid overlap in the detection of component segments. Such designs are: Yang, J. High Resolut. Chromatogr Chromatogr. Commun., 4, 83 (1981), which discloses an on-column UV detector constructed by stripping the polymer coating of a capillary and placing the capillary in the light path of a detector. Terabe et al., Anal. Chem., id., disclose a UV detector with a 0.05 by 0.75 mm slit. Walbroehl et al., J. Chromatogr., id., disclose a 100 micron pinhole as the aperture of a UV detector cell. Spino et al., J. Lig. Chromatogr., 10, 1603 (1987) disclose a detector cell fabricated by glueing a capillary and two razor blades onto a cell block, producing an aperture about 6 mm by the capillary inner diameter. Kientz et al., J. High Resolut. Chromatogr. Chromatogr. Comm., 11, 294 (1988) disclose a cell aperture made by drilling a 0.4 mm diameter hole in the outer holder of the capillary. Foret et al., Electrophoresis, 7, 430 (1986) disclose an on-column detector fabricated from optical fibers. None of the disclosed devices use aperture dimensions that maximize signal to noise ratio and at the same time prevent overlap in the detection of separated sample components.
Optimum detector performance is a function of three important aspects of the design of on-column UV detector cell apertures for CZE. First, light should only pass through the inner diameter of the capillary. When a large amount of light passes through the rim of the capillary, the signal becomes very sensitive to the refractive index changes of the solution as well as the distance between the capillary and the photodetector, and the signal to noise ratio and linear range of detection will be reduced. Second, the dimension of the aperture corresponding to the portion of the capillary segment selected for detection should be minimized to prevent overlap in the detection of separated component segments and should be adjustable to meet different detection requirements that vary with the samples to be separated. Finally, installation and removal of capillaries should be convenient and accurate.
A photodetector cell aperture capable of meeting these requirements would be highly desirable.